Methylated CpG Island Recovery Assay Research Articles

Effects of tobacco compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the expression of epigenetically regulated genes in lung carcinogenesis

ABSTRACT Cigarette smoking is a major cause of lung cancer. Although tobacco smoking-induced genotoxicity has been well established, there is apparent lack of abundance functional epigenetic effects reported On cigarette smoke-induced lung carcinogenesis. The aim of this study was to determine effects of intratracheal administration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) utilizing target gene expression DNA methylation patterns in lung tissues of mice following twice weekly for 8 weeks treatment. An unbiased approach where genomic regions was undertaken to assess early methylation changes within mouse pulmonary tissues. A methylated-CpG island recovery assay (MIRA) was performed to map the DNA methylome in lung tissues, with the position of methylated DNA determined using a Genome Analyzer (MIRA-SEQ). Alterations in epigenetic-regulated target genes were confirmed with quantitative reverse transcription-PCR, which revealed 35 differentially hypermethylated genes including Cdkn1C, Hsf4, Hnf1a, Cdx1, and Hoxa5 and 30 differentially hypomethylated genes including Ddx4, Piwi1, Mdm2, and Pce1 in NNK-exposed lung tissue compared with controls. The main pathway of these genes for mediating biological information was analyzed using the Kyoto Encyclopedia of Genes and Genomes database. Among them, Rssf1 and Mdm2 were closely associated with NNK-induced lung carcinogenesis. Taken together, our data provide valuable resources for detecting cigarette smoke-induced lung carcinogenesis.

MP45-01 ANDROGENIC TO ESTROGENIC SWITCH IN THE PROSTATE GLAND OF OVERWEIGHT PATIENT

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology1 Apr 2018MP45-01 ANDROGENIC TO ESTROGENIC SWITCH IN THE PROSTATE GLAND OF OVERWEIGHT PATIENT Zongwei Wang, Libing Hu, Shulin Wu, Shahin Tabetabaei, Chin-Lee Wu, and Aria Olumi Zongwei WangZongwei Wang More articles by this author , Libing HuLibing Hu More articles by this author , Shulin WuShulin Wu More articles by this author , Shahin TabetabaeiShahin Tabetabaei More articles by this author , Chin-Lee WuChin-Lee Wu More articles by this author , and Aria OlumiAria Olumi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1440AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The steroid 5-α reductase type 2 (SRD5A2) is critical for prostatic development and growth. Strategies to block SRD5A2 using 5-α reductase inhibitors (5ARI) remain a mainstay in the treatment of benign prostatic hyperplasia (BPH). However, one-third of men are resistant to 5ARI therapies. We previously showed that body mass index (BMI) correlates with increased SRD5A2 gene promoter methylation and decreased protein expression in men with symptomatic BPH. We have demonstrated that there is an androgenic to estrogenic switch when SRD5A2 is absent in the prostate gland. Here we wished to identify whether BMI is associated with the androgenic to estrogenic switch in human prostate tissue. METHODS Prostate specimens were collected from 35 patients who underwent transurethral resection of the prostate for symptomatic BPH at Massachusetts General Hospital. Medical records were reviewed to retrospectively collect clinical and pathological data. Patients were categorized by BMI as lean (less than 25 kg/m2), and overweight (25 kg/m2 or greater). Use and duration of α-blockers and/or 5ARIs was assessed. Methylation of SRD5A2 promoter was assessed using Methylated CpG Island Recovery Assay (MIRA). Prostatic levels of testosterone, dihydrotestosterone and estradiol were measured by HPLC-MS. RESULTS We found that BMI was significantly correlated with methylation of SRD5A2 gene promoter (p<0.05) and absence of SRD5A2 protein expression. Higher BMI was associated with higher prostatic estradiol levels (p<0.05). IIEF-5 score, International Index of Erectile Function Questionnaire score, was negatively associated with BMI (p<0.05). Treatment with 5ARIs dramatically increased the level of prostate testosterone levels and testosterone/estradiol ratio in the prostate specimens (p<0.01, p=0.01, respectively), and decreased the level of dihydrotestosterone (p<0.05). CONCLUSIONS Our study demonstrates for the first time that there is an androgenic to estrogenic switch in the prostate glands of overweight patients. Associated with body weight, somatic epigenetic silencing of SRD5A2 changes the prostatic hormonal milieu, and may modulate prostatic homeostasis and growth. Targeting the estrogenic signaling may serve as an effective treatment strategy in subset of overweight BPH patients. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e597 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Zongwei Wang More articles by this author Libing Hu More articles by this author Shulin Wu More articles by this author Shahin Tabetabaei More articles by this author Chin-Lee Wu More articles by this author Aria Olumi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A Versatile Assay for Detection of Aberrant DNA Methylation in Bladder Cancer.

Urothelial carcinoma of the bladder is one of the most common malignancies in the industrialized world, mainly caused by smoking and occupational exposure to chemicals. The favorable prognosis of early stage bladder cancer underscores the importance of early detection for the treatment of this disease. The high recurrence rate of this malignancy also highlights the need for close post-diagnosis monitoring of bladder cancer patients. As for other malignancies, aberrant DNA methylation has been shown to play a crucial role in the initiation and progression of bladder cancer, and thus holds great promise as a diagnostic and prognostic biological marker. Here, we describe a protocol for a versatile DNA methylation enrichment method, the Methylated CpG Island Recovery Assay (MIRA), which enables analysis of the DNA methylation status in individual genes or across the entire genome. MIRA is based on the ability of the methyl-binding domain (MBD) proteins, the MBD2B/MBD3L1 complex, to specifically bind methylated CpG dinucleotides. This easy-to-perform method can be used to analyze the methylome of bladder cancer or urothelial cells shed in the urine to elucidate the evolution of bladder carcinogenesis and/or identify epigenetic signatures of chemicals known to cause this malignancy.

DNA Methylation Changes in Tbx3 in a Mouse Model Exposed to Polybrominated Diphenyl Ethers.

DE-71, a commercial mixture of polybrominated diphenyl ethers widely used in flame retardants, is a pervasive environmental contaminant due to its continuing release from waste material and its long half-life in humans. Although the genotoxic potential of DE-71 appears to be low based on bacterial mutagenicity, it remains a public health concern due to its reported involvement in tumor development. Molecular mechanisms by which DE-71 influences tumor incidence or progression remain understudied. We used liver carcinoma tissue from mice exposed to DE-71 to test the hypothesis that epigenetic alterations consistent with tumor development, specifically DNA methylation, result from long-term DE-71 exposure. We profiled DNA methylation status using the methylated-CpG island recovery assay coupled with microarray analysis of hepatocellular carcinoma DNA from animals exposed to DE-71. DE-71 exposure had little impact on global DNA methylation. However, we detected gene body-specific hypomethylation within the Tbx3 locus, a transcription factor important in liver tumorigenesis and in embryonic and cancer stem cell proliferation. This nonpromoter hypomethylation was accompanied by upregulation of Tbx3 mRNA and protein and by alterations in downstream cell cycle-associated marker expression. Thus, exposure to DE-71 may facilitate tumor development by inducing epigenetic programs that favor expansion of progenitor cell populations.

Hypermethylation of antisense long noncoding RNAs in acute lymphoblastic leukemia.

Long noncoding RNAs serve critical regulatory functions highly specific for a tissue and its developmental stage. Antisense long ncRNA (AS-lncRNA) methylation changes in acute lymphoblastic leukemia (ALL) versus normal pre-B-cell lymphoblasts were evaluated to identify potential differential methylation in this group of genes. The methylome of ALL and normal lymphoblasts was examined by the methylated CpG island recovery assay followed by NGS. The potential effect of trans regulation by AS-lncRNA through DNA/RNA binding is significant as sequence alignment analysis of the 25 most differentially methylated AS-lncRNAs revealed 368 genes containing highly similar sequences with a median nucleotide identity of 90.8% and binding span of 122 base pairs. Regulation of biological processes and anatomical structure development were over represented. ALL classification schemes based on AS-lncRNA methylation can provide new insights into its pathogenesis and treatment.

Genome-Wide DNA Methylation Profiling in Diabetogenic and Regulatory T Cells from NOD Mice

A well-balanced population of pathogenic T (Tpath) and regulatory T (Treg) cells is important for maintaining immune tolerance and preventing autoimmune diseases. However, the molecular mechanisms modulating Tpath or Treg functional differentiation remain largely unclear. Using methylated CpG island recovery assay (MIRA), we have analyzed DNA methylation profiling in one Tpath, two Tregs and one control T cell line (Tctrl). We have identified a total of 2204 different methylation peaks in the four cells and ~1300 peaks in each cell. Analyses of autosomes in Tpath showed relatively lower methylation density than those in Tregs, except for Chromsome 19 that has similar methylation density. Furthermore, 63 hypermethylation and 268 hypomethylation peaks were uniquely found in Tpath, compared to 323 hypermethylation and 30 hypomethylation unique peaks in Tregs. However, the methylation density of Chromosome X in Tpath is significantly higher than that in Tregs. In summary, opposite epigenetic mechanisms are likely involved in methylation of genes on autosomes or Chromosome X in Tpaths and Tregs, thus contributing to the functional differentiation/specification between Tpaths and Tregs.

Epigenome-wide analysis links SMAD3 methylation at birth to asthma in children of asthmatic mothers

The timing and mechanisms of asthma inception remain imprecisely defined. Although epigenetic mechanisms likely contribute to asthma pathogenesis, little is known about their role in asthma inception. We sought to assess whether the trajectory to asthma begins already at birth and whether epigenetic mechanisms, specifically DNA methylation, contribute to asthma inception. We used the Methylated CpG Island Recovery Assay chip to survey DNA methylation in cord blood mononuclear cells from 36 children (18 nonasthmatic and 18 asthmatic subjects by age 9years) from the Infant Immune Study (IIS), an unselected birth cohort closely monitored for asthma for a decade. SMAD3 methylation in IIS (n=60) and in 2 replication cohorts (the Manchester Asthma and Allergy Study [n=30] and the Childhood Origins of Asthma Study [n=28]) was analyzed by using bisulfite sequencing or Illumina 450K arrays. Cord blood mononuclear cell-derived IL-1β levels were measured by means of ELISA. Neonatal immune cells harbored 589 differentially methylated regions that distinguished IIS children who did and did not have asthma by age 9years. In all 3 cohorts methylation in SMAD3, the most connected node within the network of asthma-associated, differentially methylated regions, was selectively increased in asthmatic children of asthmatic mothers and was associated with childhood asthma risk. Moreover, SMAD3 methylation in IIS neonates with maternal asthma was strongly and positively associated with neonatal production of IL-1β, an innate inflammatory mediator. The trajectory to childhood asthma begins at birth and involves epigenetic modifications in immunoregulatory and proinflammatory pathways. Maternal asthma influences epigenetic mechanisms that contribute to the inception of this trajectory.

DNA methylome changes by estradiol benzoate and bisphenol A links early-life environmental exposures to prostate cancer risk

ABSTRACTDevelopmental exposure to endocrine-disrupting chemicals (EDCs), 17β-estradiol-3-benzoate (EB) and bisphenol A (BPA), increases susceptibility to prostate cancer (PCa) in rodent models. Here, we used the methylated-CpG island recovery assay (MIRA)-assisted genomic tiling and CpG island arrays to identify treatment-associated methylome changes in the postnatal day (PND)90 dorsal prostate tissues of Sprague-Dawley rats neonatally (PND1, 3, and 5) treated with 25 µg/pup or 2,500 µg EB/kg body weight (BW) or 0.1 µg BPA/pup or 10 µg BPA/kg BW. We identified 111 EB-associated and 86 BPA-associated genes, with 20 in common, that have significant differentially methylated regions. Pathway analysis revealed cancer as the top common disease pathway. Bisulfite sequencing validated the differential methylation patterns observed by array analysis in 15 identified candidate genes. The methylation status of 7 (Pitx3, Wnt10b, Paqr4, Sox2, Chst14, Tpd52, Creb3l4) of these 15 genes exhibited an inverse correlation with gene expression in tissue samples. Cell-based assays, using 5-aza-cytidine-treated normal (NbE-1) and cancerous (AIT) rat prostate cells, added evidence of DNA methylation-mediated gene expression of 6 genes (exception: Paqr4). Functional connectivity of these genes was linked to embryonic stem cell pluripotency. Furthermore, clustering analyses using the dataset from The Cancer Genome Atlas revealed that expression of this set of 7 genes was associated with recurrence-free survival of PCa patients. In conclusion, our study reveals that gene-specific promoter methylation changes, resulting from early-life EDC exposure in the rat, may serve as predictive epigenetic biomarkers of PCa recurrence, and raises the possibility that such exposure may impact human disease.

Hypomethylation of human-specific family of LINE-1 retrotransposons in circulating DNA of lung cancer patients

Circulating DNA has recently gained attention as a fast and non-invasive way to assess tumor biomarkers. Since hypomethylation of LINE-1 repetitive elements was described as one of the key hallmarks of tumorigenesis, we aimed to establish whether the methylation level of LINE-1 retrotransposons changes in cell-surface-bound fraction of circulating DNA (csbDNA) of lung cancer patients. Methylated CpG Island Recovery Assay (MIRA) coupled to qPCR-based quantitation was performed to assess integral methylation level of LINE-1 promoters in csbDNA of non-small cell lung cancer patients (n=56) and healthy controls (n=44). Deep sequencing of amplicons revealed that hypomethylation of LINE-1 promoters in csbDNA of lung cancer patients is more pronounced for the human-specific L1Hs family. Statistical analysis demonstrates significant difference in LINE-1 promoter methylation index between cancer patients and healthy individuals (ROC-curve analysis: n=100, AUC=0.69, p=0.0012) and supports the feasibility of MIRA as a promising non-invasive approach.

Regulation of steroid-5-alpha-reductase 2 (SRD5A2) in human prostate by epigenetic modifications.

204 Background: 5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth. We have found that many aging prostate tissues do not express the enzyme. We previously showed that expression of SRD5A2 is not static and epigenetic modulations by DNA methyltransferase and pro-inflammatory cytokines play important roles in silencing of SRD5A2. Here we wished to define the methylation pattern and identify specific CpG dinucleotides that are methylated in the SRD5A2promoter region. Methods: Ninety six prostate samples from patients were obtained by transurethral resection of prostate (TURP). Methylation of SRD5A2 promoter was assessed using Methylated CpG Island Recovery Assay (MIRA). Bisulfide conversion of Genomic DNA was performed using Epimark bisulfide conversion kit. Primers for bisulfide sequencing were designed using MethPrimer software and bisulfide sequencing was performed using 3730XL DNA Sequencers. Binding of histone demethylase to promoter DNA was analyzed by ChiP assay. Results: We randomly selected 3 TURP samples with methylated SRD5A2 promoters and 3 TURP samples with un-methylated SRD5A2 promoters, and then bisulfide conversion and sequencing were performed to verify the methylation pattern of CpG dinucleotides on SRD5A2 promoter. We found that bisulfide sequencing analysis of SRD5A2 promoter was consistent with MIRA analysis. To further analyze the mediators of SRD5A2 promoter region, we show that TNF-alpha up-regulates expression of Snail protein, which is a central regulator of both DNA methylation and histone methylation, and increases the binding of H3K9me to SRD5A2promoter region, but not H3K27me or H4K20me. Conclusions: Methylation of SRD5A2 promoter, which is regulated by DNMT1, proinflammatory cytokines and the histone methylase, H3K9me, accounts for suppression of SRD5A2 in many adult human prostate tissues. Bisulfide conversion and sequencing confirm and focus the specified CpG dinucleotides that are methylated enabling us to evaluate mechanisms and patterns of SRD5A2 promoter methylation in order to study the functional significance of SRD5A2 methylation. Grant support: NIH/R01 DK091353.

Integrated methylome and transcriptome analysis reveals novel regulatory elements in pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children under the age of 15. In addition to genetic aberrations, epigenetic modifications such as DNA methylation are altered in cancer and impact gene expression. To identify epigenetic alterations in ALL, genome-wide methylation profiles were generated using the methylated CpG island recovery assay followed by next-generation sequencing. More than 25,000 differentially methylated regions (DMR) were observed in ALL patients with ∼90% present within intronic or intergenic regions. To determine the regulatory potential of the DMR, whole-transcriptome analysis was performed and integrated with methylation data. Aberrant promoter methylation was associated with the altered expression of genes involved in transcriptional regulation, apoptosis, and proliferation. Novel enhancer-like sequences were identified within intronic and intergenic DMR. Aberrant methylation in these regions was associated with the altered expression of neighboring genes involved in cell cycle processes, lymphocyte activation and apoptosis. These genes include potential epi-driver genes, such as SYNE1, PTPRS, PAWR, HDAC9, RGCC, MCOLN2, LYN, TRAF3, FLT1, and MELK, which may provide a selective advantage to leukemic cells. In addition, the differential expression of epigenetic modifier genes, pseudogenes, and non-coding RNAs was also observed accentuating the role of erroneous epigenetic gene regulation in ALL.

Abstract 1052: Variable expression of 5-alpha reductase 2 in the aging adult prostate is regulated by DNA methylation

Abstract BACKGROUND: 5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth, is utilized as an inhibitory target by finasteride for patients with bladder outlet obstrution secondary to BPH. However, we have found that many aging benign prostate tissues do not express the enzyme. Since the SRD5A2 promoter contains a CpG island, we hypothesized that somatic methylation of the promoter would be regulated by DNA methyltransferases leading to suppression of SRD5A2. METHODS: Benign prostatic tissues from wild-type mice at 3, 6 and 12 months of age were used. In addition, 96 prostate samples from human male patients who were treated by TURP for bladder outlet obstruction secondary to BPH were used. Methylation of SRD5A2 promoter was assessed using Methylated CpG Island Recovery Assay (MIRA). DNMT1 siRNA and 5-AZA-C were used to determine the methylation status of SRD5A2 in benign prostatic cell line, BPH-1. To determine the effect of CpG methylation, SRD5A2 promoter-luciferase constructs were methylated in vitro using M.SssI methylase. Statistical analyses were performed with JMP Pro version 11 (SAS Institute Inc., Cary, NC). RESULTS: We analyzed 96 human BPH samples from transurethral resection of prostate (TURP) surgeries and found that absence of SRD5A2 was closely associated with hypermethylation of the SRD5A2 promoter region. We found that methylation of SRD5A2 was regulated by DNA methytransferase 1 (DNMT1) and the cytokines, TNF-alpha, NF-κB and IL-6, regulated DNMT1protein expression and thereby indirectly affected SRD5A2 promoter methylation and gene expression. Suppression of cytokines inhibited activity of DNMT1, while over-expression of p65 or treatment with TNF-alpha or IL-6 up-regulated DNMT1. In addition, we found that methylation of the SRD5A2 promoter and concomitant decrease in protein expression were closely associated with patient's increasing age (P&amp;lt;0.05). Finally, in murine prostate tissues, cytokine levels, DNMT1 and global methylation were all increased in older mice. Induction of inflammation with lipopolysaccharide (LPS) stimulated the TNFR1/NF-κB/IL-6/DNMT1 pathway, leading to hypermethylation of the SRD5A2 promoter and silencing of SRD5A2, while treatment with both LPS and TNF-alpha inhibitor reversed this pathway and reactivated SRD5A2. CONCLUSIONS: Expression of SRD5A2 is not static and ubiquitous in benign adult prostate tissues. We show that SRD5A2 expression is lacking in many benign human adult prostate tissues. Methylation of SRD5A2 promoter region, which is regulated by DNMT1, accounts for absence of SRD5A2 expression in many adult human prostate tissues. Methylation and expression of SRD5A2 may be used as a gene signature to tailor therapies for more effective treatment of prostatic diseases. Citation Format: Ge Rongbin, Zongwei Wang, Seth Bechis, Alexander Otsetov, Shengyu Hua, Shulin Wu, Chin-Lee Wu, Shahin Tabatabaei, Aria Olumi. Variable expression of 5-alpha reductase 2 in the aging adult prostate is regulated by DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1052. doi:10.1158/1538-7445.AM2015-1052

Abstract 3840: A new prognostic marker: interferon regulatory factor 6 in renal cell carcinoma

Abstract According to our previous methylated-CpG island recovery assay (MIRA) and RNA expression array findings, the results indicated methylated status of Interferon regulatory factor 6 (IRF6) could be observed in most of renal cell carcinoma (RCC) cases (40.6%), and presented a negative correlation with gene expression, clear cell type of RCCs especially. As known that IRF6 is one of members of IRF family. It has been described might play an important role in epidermal cells, and correlated with cell proliferation and differentiation. The regulation of feedback loop between IRF6 and ΔNp63, which is known could control the mechanism of keratinocytes differentiation and palate closure, has also been noted. In addition, the tumor suppressor activity of IRF6 had been also demonstrated in squamous cell carcinoma associated with inhibition of tumor cell invasion and migration. The aim of this study is to clarify the clinical significance and role of IRF6 in RCC. We hypothesized low expression of IRF6 via DNA methylation might lead to irregular differentiation of RCC cells, and then further trigger cells proliferated out of control. In total, 105 pairs of clinical RCCs patients and eight RCC cell lines (included chromophobe type RCC98, sacomatoid type RCC52, clear cell type Caki-1, HH332, HOKN-9, RCC100, papillary type HH050 and adenocarcinoma 786-O), as well as Human Embryonic Kidney 293 cell used as control have involved in current study. Firstly, we performed real-time PCR on all cases. Secondly, 5-aza-2′deoxycytidine was treated on all type of RCC cell lines; it is a way of de-methylated DNA. Western blot detections were then to confirm whether the expression level of IRF6 restored in RCC cells. The IRF6 gene expressed levels differ from normal and RCC tissues were applied by the paired- T test, and shown by -ΔCT. Generally, the variant and lower level gene expression of the IRF6 could be observed from all different type of RCC cell lines, except RCC98 and RCC100 cells. After treated with 5-aza-2′deoxycytidine on RCC cell lines, the obvious expression of IRF6 was restored in all clear cell RCC cell lines. The mean - ΔCT of normal tissue was -8.0, and RCC tissue was -11.5, significantly IRF6 expressed levels differ from normal and tumor tissues could be noted (p = 0.013). The IRF6 gene was usually expressed lower level in RCC due to methylation. Our findings demonstrated the expression of IRF6 could be easily resorted by de-methylated in RCC cells, and it might further inhibit cell proliferation in the tumoral progression, and might consider as a prognostic marker to predict patients’ outcomes in RCC. However, the further cell viability and correlation with the clinical information should be further analyzed in the future. Citation Format: Chih-Yang Wei, Ying-Tzu Chen, See-Tong Pang, Wen-Hui Weng. A new prognostic marker: interferon regulatory factor 6 in renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3840. doi:10.1158/1538-7445.AM2015-3840

MIRA-seq for DNA methylation analysis of CpG islands.

Aim:To develop a reliable method for whole genome analysis of DNA methylation.Materials & methods:Genome-scale analysis of DNA methylation includes affinity-based approaches such as enrichment using methyl-CpG-binding proteins. One of these methods, the methylated-CpG island recovery assay (MIRA), is based on the high affinity of the MBD2b-MBD3L1 complex for CpG-methylated DNA. Here we provide a detailed description of MIRA and combine it with next generation sequencing platforms (MIRA-seq).Results:We assessed the performance of MIRA-seq and compared the data with whole genome bisulfite sequencing.Conclusion:MIRA-seq is a reliable, genome-scale DNA methylation analysis platform for scoring DNA methylation differences at CpG-rich genomic regions. The method is not limited by primer or probe design and is cost effective.

Age dependent changes in the LPS induced transcriptome of bovine dermal fibroblasts occurs without major changes in the methylome.

BackgroundBy comparing fibroblasts collected from animals at 5-months or 16-months of age we have previously found that the cultures from older animals produce much more IL-8 in response to lipopolysaccharide (LPS) stimulation. We now expand this finding by examining whole transcriptome differences in the LPS response between cultures from the same animals at different ages, and also investigate the contribution of DNA methylation to the epigenetic basis for the age-dependent increases in responsiveness.ResultsAge-dependent differences in IL-8 production by fibroblasts in response to LPS exposure for 24 h were abolished by pretreatment of cultures with a DNA demethylation agent, 5-aza-2′deoxycytidine (AZA). RNA-Seq analysis of fibroblasts collected from the same individuals at either 5 or 16 months of age and exposed in parallel to LPS for 0, 2, and 8 h revealed a robust response to LPS that was much greater in the cultures from older animals. Pro-inflammatory genes including IL-8, IL-6, TNF-α, and CCL20 (among many other immune associated genes), were more highly expressed (FDR < 0.05) in the 16-month old cultures following LPS exposure. Methylated CpG island recovery assay sequencing (MIRA-Seq) revealed numerous methylation peaks spread across the genome, combined with an overall hypomethylation of gene promoter regions, and a remarkable similarity, except for 20 regions along the genome, between the fibroblasts collected at the two ages from the same animals.ConclusionsThe fibroblast pro-inflammatory response to LPS increases dramatically from 5 to 16 months of age within individual animals. A better understanding of the mechanisms underlying this process could illuminate the physiological processes by which the innate immune response develops and possibly individual variation in innate immune response arises. In addition, although relatively unchanged by age, our data presents a general overview of the bovine fibroblast methylome as a guide for future studies in cattle epigenetics utilizing this cell type.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1223-z) contains supplementary material, which is available to authorized users.

Genome-wide DNA methylation analysis in precursor B-cells

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182–200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.

Abstract 2313: Dynamic expression of 5-alpha reductase 2 in aging prostate is regulated by DNA methyltransferase 1

Abstract BACKGROUND: 5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth, is utilized as an inhibitory target by Finasteride for patients with BPH. However, we have found that many aging benign prostate tissues do not express the enzyme. Since the SRD5A2 promoter contains a CpG island, we hypothesized that somatic methylation of the promoter may account for absence of SRD5A2 expression. METHODS: Benign prostatic tissues from wild-type mice at 3, 6 and 12 months of age were used. In addition, prostate samples from human male patients who were treated by TURP for bladder outlet obstruction secondary to BPH were used. Methylation of SRD5A2 promoter was assessed using Methylated CpG Island Recovery Assay (MIRA). DNMT1 siRNA and 5-AZA-C were used to determine the methylation status of SRD5A2 in benign prostatic cells (BPH-1). SRD5A2 promoter-luciferase constructs were methylated in vitro using M.SssI methylase. RESULTS: Benign prostatic tissues from wild-type mice of different ages (3 months, 6 months and 12 months, n=6 per group) were harvested. We found that expression of SRD5A2 and DNMT1, 3a, 3b were not significantly changed in mice at the age of 3 and 6 months. However, 33% of mice at age of 12 months (2/6) had low levels of SRD5A2 with concomitant high DNMT1 protein levels. Consistent with reduced expression of SRD5A2 and increased DNMT1, the methylation of SRD5A2 promoter was increased as assayed by MIRA. Moreover, silencing DNMT1 with siRNA or exposure of BPH-1 cells to 5-AZA-C led to re-expression of SRD5A2. We used SRD5A2 promoter-luciferase constructs that were methylated in vitro using M.SssI methylase. When the methylated SRD5A2 promoter constructs were ectopically expressed in BPH-1 cells, we found that methylation of the SRD5A2 promoter caused reduction of luciferase expression by 80%, suggesting that methylation of the SRD5A2 promoter regulates the expression of SRD5A2. Since age-associated increased IL-6 has been shown to regulate DNMT1, we next evaluated expression of IL-6 in aging prostate tissue. We found that expression of IL-6 and its downstream molecule, phosphorylated-STAT3, correlated with expression of DNMT1. Concurrent with our animal and in-vitro studies, we analyzed 80 benign human prostate samples from TURPs and found that 24/80 (30%) of did not express the SRD5A2 protein with concurrent methylation of SRD5A2 promoter, suggesting that methylation and absence of of SRD5A2 protein expression are closely linked (p=0.0006, Fisher's exact test). This data suggests that expression of SRD5A2 is highly variable in human adult prostate tissue. CONCLUSIONS: Expression of SRD5A2 in aging prostates is a dynamic process that is regulated by methylation of SRD5A2 promoter. DNMT1 regulates the methylation and expression of SRD5A2. Dynamic and variable expression of SRD5A2 has implications for management of BPH and chemopreventive strategies for prostate cancer. Citation Format: Ge Rongbin, Zongwei Wang, Chin-Lee Wu, Shahin Tabatabaei, Aria Olumi. Dynamic expression of 5-alpha reductase 2 in aging prostate is regulated by DNA methyltransferase 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2313. doi:10.1158/1538-7445.AM2014-2313

MIRA analysis of RARβ2 gene methylation in DNA circulating in the blood in lung cancer.

Analysis of DNA epigenetic mutations in the blood circulating DNA is a prospective trend for creation of noninvasive methods for the diagnosis and treatment efficiency monitoring in cancer. The methylation status of target genes in circulating DNA was evaluated by methods based on preliminary bisulfite conversion of DNA. We used a different approach based on selection of hypermethylated sequences of circulating DNA by means of DNA-methyl-binding protein (methylated CpG island recovery assay, MIRA). Methylation was evaluated for RARβ2 tumor suppression gene in circulating DNA in lung cancer and a trend was detected to higher methylation of this gene in the patients in comparison with healthy donors.

De Novo DNA Methylation in the Male Germ Line Occurs by Default but Is Excluded at Sites of H3K4 Methylation

To understand what dictates the emerging patterns of de novo DNA methylation in the male germline, we mapped DNA methylation, chromatin, and transcription changes in purified fetal mouse germ cells by using methylated CpG island recovery assay (MIRA)-chip, chromatin immunoprecipitation (ChIP)-chip, and strand-specific RNA deep sequencing, respectively. Global de novo methylation occurred by default in prospermatogonia without any apparent trigger from preexisting repressive chromatin marks but was preceded by broad, low-level transcription along the chromosomes, including the four known paternally imprinted differentially methylated regions (DMRs). Default methylation was excluded only at precisely aligned constitutive or emerging peaks of H3K4me2, including most CpG islands and some intracisternal A particles (IAPs). Similarly, each maternally imprinted DMR was protected from default DNAmethylation among highly methylated DNA by an H3K4me2 peak and transcription initiation at least in one strand. Our results suggest that the pattern of de novo DNA methylation in prospermatogonia is dictated by opposing actions of broad, low-level transcription and dynamic patterns of active chromatin.

Abstract 2976: Dynamic expression of 5-alpha reductase 2 in aging prostate is regulated by DNA methyltransferase 1.

Abstract Introduction: 5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth, is utilized as an inhibitory target by Finasteride for patients with BPH. However, we have found that many aging benign prostate tissues do not express the enzyme. Since the SRD5A2 promoter region contains a CpG island, we hypothesized that somatic methylation of the promoter region may account for absence of SRD5A2 expression and DNA methyltransferases may play a role in silencing SRD5A2 expression. Methods: Benign prostatic tissues from wild-type mice at 3, 6 and 12 months of age were used. Expression of SRD5A2 and DNA methyltransferases were analyzed by Western blot and quantitative RT-PCR. Methylation of SRD5A2 promoter region was assessed using Methylated CpG Island Recovery Assay (MIRA). DNMT1 siRNA and 5-AZA-C were used to determine the methylation status of SRD5A2 in benign prostatic cells (BPH-1). To determine the effect of CpG methylation, SDR5A2 promoter-luciferase contrsucts were methylated in vitro using M.SssI methylase. IL-6 expression was analyzed using ELISA. Results: Benign prostatic tissues from wild-type mice of different ages (n=5 per group) were harvested. We found that expression of SRD5A2 and DNMT 1, 3a, 3b were not significantly changed in mice at the age of 3 and 6 months. However, 40% of mice at age of 12 months (2/5) demonstrated low SRD5A2 expression and high DNMT1 expression. Expression of DNMT3a and 3b were not altered. Consistently, the methylation status of SRD5A2 promoter was confirmed by Methylated CpG Island Recovery Assay (MIRA). To study the role of DNMT1 in silencing of SRD5A2, we silenced DNMT1 using siRNA in BPH-1 cells. Silencing DNMT1 led to re-expression of SRD5A2. Similarly, exposure of cells to 5-AZA-C led to re-expression of SDR5A2. The 5-AZA-C only led to increased expression of DNMT1 and not not DNMT3a or DNMT3b in BPH-1 cells. We used SDR5A2 promoter-luciferase contrsucts that were methylated in vitro using M.SssI methylase technique. When the methylated SDR5A2 promoter constructs were ectopically expressed in BPH-1 cells, we found that methylation of the SDR5A2 promoter caused reduction of luciferase gene expression by 80%, suggesting that methylation of the SDR5A2 promoter regulates the expression of SDR5A2. Since age-associated increased IL-6 has been shown to regulate DNMT-1, we next evaluated expression of IL-6 in aging prostate tissue. We found that expression of IL-6 was positively correlated with expression DNMT-1 in benign prostatic tissues. Conclusions: Expression of SRD5A2 in aging prostates is dynamic. Absence of SRD5A2 expression is associated with methylation of the gene's promoter region, which is regulated by DNMT1 and not DNMT3a or DNMT3b. Dynamic and absence of SRD5A2 has implications for management of BPH and chemopreventive strategies for prostate cancer. Citation Format: Rongbin Ge, Zongwei Wang, Chin-Lee Wu, Shahin Tabatabaei, Aria F. Olumi. Dynamic expression of 5-alpha reductase 2 in aging prostate is regulated by DNA methyltransferase 1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2976. doi:10.1158/1538-7445.AM2013-2976